Active or mature proteins are usually present in very low amounts in living organisms. Therefore, their pro-proteins or pro-enzymes are preferably activated in vitro by contacting them with activation enzymes (e.g. proteases). Pro-proteins (or protein precursors) are inactive proteins that become active by one or more posttranslational modifications and, in particular, by the cleavage of a pro-peptide from the pro-protein. Examples of pro-proteins include, for example, pro-insulin, prothrombin, pro-von Willebrand Factor (pro-VWF), and the like.
Von Willebrand factor (VWF) is a blood glycoprotein involved in coagulation. VWF is deficient or defective in von Willebrand disease and is involved in a large number of other diseases, including thrombotic thrombocytopenic purpura, Heyde's syndrome, and possibly hemolytic-uremic syndrome. VWF is a glycoprotein circulating in plasma as a series of multimers ranging in size from about 500 to 20,000 kD. Multimeric forms of VWF are composed of 250 kD polypeptide subunits linked together by disulfide bonds. VWF mediates the initial platelet adhesion to the sub-endothelium of the damaged vessel wall, and it is believed that only the larger multimers of VWF exhibit hemostatic activity. VWF multimers having large molecular masses are stored in the Weibel-Pallade bodies of endothelial cells and are liberated upon stimulation. Liberated VWF is then further processed by plasma proteases to result in low molecular weight forms of VWF.
In humans the removal of the pro-peptide is almost complete, whereas, in mammalian cell lines with a high level of recombinant VWF expression, this process is not very efficient. Therefore, cell culture supernatants from such recombinant cell lines usually comprise a mixture of mature VWF and VWF precursors, like pro-VWF. In order to obtain mature VWF, it is therefore necessary to convert the VWF precursors, in particular pro-VWF, into mature VWF. This process is usually achieved by cleaving the pro-peptide with a protease.
Current conventional methods produce mature VWF by either incubating its pro-form with proteases in a liquid phase, whereby the maturation itself (i.e., the cleavage of the pro-peptide from the pro-protein) occurs in an unbound state in free solution, or as described, for example, in WO 00/49047, by immobilizing the protease on a solid carrier, which is contacted and incubated with a preparation comprising pro-VWF (see e.g. WO 00/49047). However, these methods comprise various disadvantages over the methods according to the present invention.
Industrially, VWF and, in particular, recombinant VWF (rVWF) is synthesized and expressed together with recombinant Factor VIII (rFVIII) in a genetically engineered Chinese Hamster ovary (CHO) cell line. The function of the co-expressed rVWF is to stabilize rFVIII in the cell culture process. rVWF is synthesized in the cell in the pro-form, containing a large pro-peptide attached to the N-terminus. Upon maturation in the endoplasmic reticulum and Golgi apparatus, the pro-peptide is cleaved by the action of the cellular protease furin and the mature protein is secreted as a homopolymer of identical subunits, consisting of dimers of the expressed protein. However, the maturation is typically incomplete, leading to a product comprising a mixture of pro-VWF and mature VWF.
Previous publications have shown that pro-VWF can be converted to mature VWF by in vitro treatment with furin or furin-like proteases (Schlokat et al., Biotechnol. Appl. Biochem. 24: 257-267, 1996; Preininger et al., Cytotechnology 30: 1-15, 1999; and EP 0775750A). In particular, EP 0775750A suggests the co-expression of furin and VWF recombinantly so that the maturation of VWF may occur in situ.
Recombinant furin (rFurin) transforms pro-rVWF (pro-recombinant von Willebrand factor) to rVWF by cleaving the Arg741-Ser742 peptide bond. This maturation step is part of a rVWF production process for the treatment of von Willebrand Disease Type B and part of the manufacturing process for recombinant Factor VIII-half life (rFVIII-HL). Furin belongs to the family of the pro-protein convertases and is dependent on calcium (Ca2+). Furin specifically cleaves the C-terminal peptide bond of arginine within a specific sequence, containing arginine at positions −1 and −4. This sequence can be found in numerous human proteins, showing that furin plays a major role in the maturation of a number of human pro-proteins.
The production of activated proteins is of high clinical and diagnostic importance. For example, active or mature proteins, like mature VWF, may be used to control blood coagulation. The present invention provides improved recombinant furin (rFurin) which is substantially animal protein-free rFurin for the subsequent production of activated proteins. More specifically, the present invention provides substantially animal protein-free rFurin for transforming pro-VWF into mature VWF.